Compounds enhancing human sperm motility identified using a high-throughput phenotypic screening platform

Abstract

STUDY QUESTION Can a high-throughput screening (HTS) platform facilitate male fertility drug discovery? SUMMARY ANSWER An HTS platform identified a large number of compounds that enhanced sperm motility. WHAT IS KNOWN ALREADY Several efforts to find small molecules modulating sperm function have been performed but none have used high-throughput technology. STUDY DESIGN, SIZE, DURATION Healthy donor semen samples were used and samples were pooled (3–5 donors per pool). Primary screening was performed singly; dose–response screening was performed in duplicate (using independent donor pools). PARTICIPANTS/MATERIALS, SETTING, METHODS Spermatozoa isolated from healthy donors were prepared by density gradient centrifugation and incubated in 384-well plates with compounds (6.25 μM) to identify those compounds with enhancing effects on motility. Approximately 17 000 compounds from the libraries, ReFRAME, Prestwick, Tocris, LOPAC, CLOUD and MMV Pathogen Box, were screened. Dose–response experiments of screening hits were performed to confirm the enhancing effect on sperm motility. Experiments were performed in a university setting. MAIN RESULTS AND THE ROLE OF CHANCE From our primary single concentration screening, 105 compounds elicited an enhancing effect on sperm motility compared to dimethylsulphoxide-treated wells. Confirmed enhancing compounds were grouped based on their annotated targets/target classes. A major target class, phosphodiesterase inhibitors, were identified, in particular PDE10A inhibitors as well as number of compounds not previously known to enhance human sperm motility, such as those related to GABA signalling. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Although this approach provides data about the activity of the compound, it is only a starting point. For example, further substantive experiments are necessary to provide a more comprehensive picture of each compound’s activity, the effect on the kinetics of the cell populations and subpopulations, and their potential mechanisms of action. Compounds have been tested with prepared donor spermatozoa, incubated under non-capacitating conditions, and only incubated with compounds for a relatively short period of time. Therefore, the effect of compounds under different conditions, for example in whole semen, for longer incubation times, or using samples from patient groups, may be different and require further study. All experiments were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS This phenotypic screening assay identified a large number of compounds that increased sperm motility. In addition to furthering our understanding of human sperm function, for example identifying new avenues for discovery, we highlight potential compounds as promising start-point for a medicinal chemistry programme for potential enhancement of male fertility. Moreover, with disclosure of the results of screening, we present a substantial resource to inform further work in the field. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Bill and Melinda Gates Foundation, Scottish Funding Council and Scottish Universities Life Science Alliance. C.L.R.B. is Editor for RBMO. C.L.R.B. receives funding from Chief Scientists Office (Scotland), ESHRE and Genus PLC, consulting fees from Exscientia and lecture fees from Cooper Surgical and Ferring. S.M.d.S. is an Associate Editor of Human Reproduction, and an Associate Editor of Reproduction and Fertility. S.M.d.S. receives funding from Cooper Surgical and British Dietetic Society. No other authors declared a COI.

Sperm Cell Painting: A Mechanism Driven Approach for Drug Discovery in Human Spermatozoa

“…Progressive motility was measured using the high-throughput assay previously published in Gruber et al ., 2020 and 2022 [1, 2] in non-capacitating conditions and Non-capacitation media.…”

“…To begin to address these gaps in understanding, we have recently developed a high-throughput phenotypic screening platform in human sperm and used it to screen for compounds that either both decrease [1] and enhance [2] sperm motility. This platform presents opportunities for target-agnostic screening which can be adapted for screening of specific sperm functions (reviewed in Johnston et al ., 2021 [3]).…”

Sperm Toolbox – A selection of small molecules to study human spermatozoa

“…A key part of this effort is the development of mechanistic insight and drug candidates that affect the behaviour and properties of human spermatozoa (Balbach et al, 2023;Carlson et al, 2022;Faber et al, 2023;Hau et al, 2022). Towards this end, we have recently developed an automated live human sperm phenotypic, targetagnostic screening platform that can screen 1000s of compounds and identify positive and negative modifiers of sperm function (Gruber et al, 2020;Gruber et al, 2022). These effects can be either positive or negative and therefore starting points for either fertility treatments (including medical assisted reproductive technologies) or contraceptive development.…”

“…We tested the STB compounds in multiple functional sperm assays, for general cytotoxicity and aqueous solubility (Figure 1A). The STB has been assembled to contain previously published compounds and compounds detected in our own screening campaigns (Gruber et al, 2020;Gruber et al, 2022). In addition, we included a few compounds which have been inferred from patent applications or studies of proteins potentially relevant for sperm function (Figure 1B).…”

Sperm Toolbox—A selection of small molecules to study human spermatozoa

“…Towards this end, we have recently developed an automated live human sperm, target-agnostic, phenotypic screening platform that can profile the effects of 1000s of compounds on spermatozoa. These parameters are then used to identify modifiers of sperm function [ 8 , 9 ]. These modifiers can cause either positive or negative effects and therefore are starting points for either fertility treatments (including medical assisted reproductive technologies) or contraceptive development.…”